Double-attenuated influenza virus elicits broad protection against challenge viruses with different serotypes in swine.

Influenza A viruses (IAV) have caused seasonal epidemics and severe pandemics in humans. Novel pandemic strains as in 2009 may emerge from pigs, serving as perpetual virus reservoir. However, reliably effective vaccination has remained a key issue for humans and swine.

A Dual Motif in the Hemagglutinin of H5N1 Goose/Guangdong-Like Highly Pathogenic Avian Influenza Virus Strains Is Conserved from Their Early Evolution and Increases both Membrane Fusion pH and Virulence.

Zoonotic highly pathogenic avian influenza viruses (HPAIV) have raised serious public health concerns of a novel pandemic. These strains emerge from low-pathogenic precursors by the acquisition of a polybasic hemagglutinin (HA) cleavage site, the prime virulence determinant. However, required coadaptations of the HA early in HPAIV evolution remained uncertain.

Improved universal cloning of influenza A virus genes by LacZα-mediated blue/white selection.

Reverse genetics of influenza A viruses facilitates both basic research and vaccine development. However, efficient cloning of virus gene segments was cumbersome in established systems due to the necessary cleavage of amplicons with outside cutter restriction enzymes followed by ligation. Occasionally, virus genes may contain cleavage sites for those enzymes.

The Neuraminidase Stalk Deletion Serves as Major Virulence Determinant of H5N1 Highly Pathogenic Avian Influenza Viruses in Chicken.

Highly pathogenic avian influenza viruses (HPAIV) cause devastating losses in gallinaceous poultry world-wide and raised concerns of a novel pandemic. HPAIV develop from low-pathogenic precursors by acquisition of a polybasic HA cleavage site (HACS), the prime virulence determinant. Beside that HACS, other adaptive changes accumulate in those precursors prior to transformation into an HPAIV.

Avian influenza virus h3 hemagglutinin may enable high fitness of novel human virus reassortants.

Reassortment of influenza A virus genes enables antigenic shift resulting in the emergence of pandemic viruses with novel hemagglutinins (HA) acquired from avian strains. Here, we investigated whether historic and contemporary avian strains with different replication capacity in human cells can donate their hemagglutinin to a pandemic human virus. We performed double-infections with two avian H3 strains as HA donors and a human acceptor strain, and determined gene compositions and replication of HA reassortants in mammalian cells.

Diversifying evolution of highly pathogenic H5N1 avian influenza virus in Egypt from 2006 to 2011.

An evolutionary analysis was conducted of 354 hemagglutinin (HA) and 208 neuraminidase (NA) genes, including newly generated sequences of 5 HA and 30 NA, of Egyptian H5N1 clade 2.2.1 viruses isolated from poultry and humans.

Avian influenza A virus PB2 promotes interferon type I inducing properties of a swine strain in porcine dendritic cells.

The 2009 influenza A virus (IAV) pandemic resulted from reassortment of avian, human and swine strains probably in pigs. To elucidate the role of viral genes in host adaptation regarding innate immune responses, we focussed on the effect of genes from an avian H5N1 and a porcine H1N1 IAV on infectivity and activation of porcine GM-CSF-induced dendritic cells (DC). The highest interferon type I responses were achieved by the porcine virus reassortant containing the avian polymerase gene PB2.

Avian influenza virus hemagglutinins H2, H4, H8, and H14 support a highly pathogenic phenotype.

High-pathogenic avian influenza viruses (HPAIVs) evolve from low-pathogenic precursors specifying the HA serotypes H5 or H7 by acquisition of a polybasic HA cleavage site. As the reason for this serotype restriction has remained unclear, we aimed to distinguish between compatibility of a polybasic cleavage site with H5/H7 HA only and unique predisposition of these two serotypes for insertion mutations. To this end, we introduced a polybasic cleavage site into the HA of several low-pathogenic avian strains with serotypes H1, H2, H3, H4, H6, H8, H10, H11, H14, or H15, and rescued HA reassortants after cotransfection with the genes from either a low-pathogenic H9N2 or high-pathogenic H5N1 strain.

Influenza B virus with modified hemagglutinin cleavage site as a novel attenuated live vaccine.

Background: Both pandemic and interpandemic influenza is associated with high morbidity and mortality worldwide. Seasonal epidemics are caused by both influenza A and B virus strains that cocirculate with varying predominance and may give rise to severe illness equally. According to World Health Organization recommendations, current annual vaccines are composed of 2 type A and 1 type B virus-specific component.

H9 avian influenza reassortant with engineered polybasic cleavage site displays a highly pathogenic phenotype in chicken.

In the field, highly pathogenic avian influenza viruses (HPAIV) originate from low-pathogenic strains of the haemagglutinin (HA) serotypes H5 and H7 that have acquired a polybasic HA cleavage site. This observation suggests the presence of a cryptic virulence potential of H5 and H7 low-pathogenic avian influenza viruses (LPAIV). Among all other LPAIV, the H9N2 strains are of particular relevance as they have become widespread across many countries in several avian species and have been transmitted to humans.

Disruption of the viral polymerase complex assembly as a novel approach to attenuate influenza A virus.

To develop a novel attenuation strategy applicable to all influenza A viruses, we targeted the highly conserved protein-protein interaction of the viral polymerase subunits PA and PB1. We postulated that impaired binding between PA and PB1 would negatively affect trimeric polymerase complex formation, leading to reduced viral replication efficiency in vivo. As proof of concept, we introduced single or multiple amino acid substitutions into the protein-protein-binding domains of either PB1 or PA, or both, to decrease binding affinity and polymerase activity substantially.

Highly pathogenic H5N1 influenza viruses carry virulence determinants beyond the polybasic hemagglutinin cleavage site.

Highly pathogenic avian influenza viruses (HPAIV) originate from avirulent precursors but differ from all other influenza viruses by the presence of a polybasic cleavage site in their hemagglutinins (HA) of subtype H5 or H7. In this study, we investigated the ability of a low-pathogenic avian H5N1 strain to transform into an HPAIV. Using reverse genetics, we replaced the monobasic HA cleavage site of the low-pathogenic strain A/Teal/Germany/Wv632/2005 (H5N1) (TG05) by a polybasic motif from an HPAIV (TG05(poly)).

Simultaneous one-tube full-length amplification of the NA, NP, M, and NS genes of influenza A viruses for reverse genetics.

Reverse genetics of influenza A viruses has expedited increasingly basic research and vaccine development. Target-primed plasmid amplification using full-length PCR amplicons as inserts was established previously for strain-independent and rapid cloning of all eight influenza A virus genes. This method involves separate amplification of each viral gene using segment-specific primers.

Acquisition of a polybasic hemagglutinin cleavage site by a low-pathogenic avian influenza virus is not sufficient for immediate transformation into a highly pathogenic strain.

Highly pathogenic avian influenza viruses (HPAIV) differ from all other strains by a polybasic cleavage site in their hemagglutinin. All these HPAIV share the H5 or H7 subtype. In order to investigate whether the acquisition of a polybasic cleavage site by an avirulent avian influenza virus strain with a hemagglutinin other than H5 or H7 is sufficient for immediate transformation into an HPAIV, we adapted the hemagglutinin cleavage site of A/Duck/Ukraine/1/1963 (H3N8) to that of the HPAIV A/Chicken/Italy/8/98 (H5N2), A/Chicken/HongKong/220/97 (H5N1), or A/Chicken/Germany/R28/03 (H7N7) and generated the recombinant wild-type and cleavage site mutants.

Rapid and reliable universal cloning of influenza A virus genes by target-primed plasmid amplification.

Reverse genetics has become pivotal in influenza virus research relying on rapid generation of tailored recombinant influenza viruses. They are rescued from transfected plasmids encoding the eight influenza virus gene segments, which have been cloned using restriction endonucleases and DNA ligation. However, suitable restriction cleavage sites often are not available.